Instant Notes: Plant Biology

Section E – Physiology and regulation

E2 Methods in experimental plant science

Key Notes

Plant function and regulation was first studied by a combination of

surgical, chemical and genetic approaches. Recently, research has made

dramatic progress as a result of the techniques of molecular biology and

the study of mutants, plants in which genetic change can be linked to

change in form or function.

Mutants are generated by chemical treatment, radiation or inserting DNA

into the genome (insertional mutagenesis). Considerable information on

function and development can be gained by studying the phenotype of

mutants and characterizing the mutated gene. Mutants can be used in

almost all areas of experimental plant science, including development,

photosynthesis and metabolism, and nutrition and water transport.

Molecular techniques provide information on the regulation and function

of genes and their products. It is possible to determine and analyze the

entire DNA sequence of genes, describe how they are regulated, observe

the effects of both under- and over-expression and observe the effects of

deliberately mutating the gene. Reporter gene constructs (in which a gene

for a detectable product is fused with the gene of interest) have been of

great value in studying function and regulation in cells and tissues.

The location of a gene (e.g. a developmental gene which has been

mutated) on a chromosome can be identified using the technique of

mapping. The crossover frequency of the gene is compared with genetic

markers of known position on the chromosome.

Transposons are mobile pieces of DNA that insert into the genome,

disrupting the gene into which they insert. When a gene disruption is

found, it can be identified and characterized by looking for the

transposon DNA and then characterizing the surrounding region.

A transferred DNA (T-DNA) containing a selectable marker (a gene

conferring a property that permits it to be readily detected) is inserted

randomly into the genome by Agrobacterium-mediated transformation.

When it inserts into a gene of developmental importance, that gene is

disrupted. The selectable marker makes identification and

characterization of that gene straightforward.

The product of mutagenesis experiments is usually an identifiable section

of DNA containing the gene of interest, with markers for its

identification. The DNA is then isolated either from a genomic library or

from a cDNA library. It can then be sequenced and the complete amino

acid sequence of the protein product determined. Comparison with data

Approaches to study

plant function

Mutants to study

form and function

Mutagenesis by

T-DNA insertion

Molecular techniques

Map-based cloning

Transposon tagging

Identifying a

cloned gene